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Development of the Metanephric Anlage of 

Chick in Allantoic Grafts 



BY 


RUTH RAND ATTERBURY 

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Submitted in partial fulfilment of the requirements for the Degree of Doctor of 
Philosophy in the Faculty of Pure Science, Columbia University 



NEW YORK 
1923 


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Reprinted from The American Journal of Anatomy, 
Volume 31, Number 4, March, 1923 























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author’s abstract of this paper issued Reprinted from The American Journal of 

by the bibliographic service, February 10 Anatomy, Vol. 31, No. 4, March, 1923 


DEVELOPMENT OF THE METANEPHRIC ANLAGE 
OF CHICK IN ALLANTOIC GRAFTS 


RUTH RAND ATTERBURY 
Department of Anatomy, Columbia University 

FIVE TEXT FIGURES AND THREE PLATES (SEVEN FIGURES) 


CONTENTS 

Introduction. 409 

Material and method. 413 

Observations. 414 

A. Structure of the metanephric anlage at the time of grafting. 414 

B. Growth of the metanephric grafts in the allantois. 416 

1. Growth of the ureteric epithelium. 416 

2. Growth and differentiation of the nephric epithelium. 419 

3. Development of the stroma. 425 

Conclusions and discussion. 427 


INTRODUCTION 

The kidney is a structure peculiarly adapted for a study of 
the much-discussed problems of cell potentialities and cell 
differentiation and frequently has been used by investigators 
for this purpose. As is known the anlage of the permanent 
kidney is twofold consisting of, 1) the ureter bud, which gives 
rise to the collecting apparatus of the definitive kidney, and, 
2) the metanephrogenic cord, a strand of apparently uniform 
mesenchymal tissue into which the ureter bud grows. 

The cells of the metanephrogenic cord are mesodermic in 
origin. They arise from the primitive segment stalks of the 
two or three most caudal somites, which lose their metameric 
arrangement, fusing together into a continuous nephrogenic 
strand or cord. In the course of its development, the metaneph¬ 
rogenic cord takes two distinct lines of differentiation. On the 
one hand it is transformed into the highly differentiated secretory 
epithelium of the renal tubules, while on the other hand it gives 
rise to the connective-tissue capsule and stroma of the kidney. 

409 











410 


RUTH RAND ATTERBURY 


Thus two zones are usually distinguished in the metanephrogenic 
strand, an inner and an outer zone. The secretory cells of the 
nephric tubules are believed to arise from the inner zone, the 
stroma cells, from the outer zone. The cells of both zones are 
mesenchymal in character, and in the early stages of develop¬ 
ment essentially similar in structure. A difference in density 
of the tissue seems to be the only morphological distinction 
between these zones. 

The mesenchyme-like tissue of the kidney anlage, abundant 
in early stages of development, diminishes gradually in amount 
with the specific differentiation of the nephric tubules. In 
later stages of development, as well as in the adult kidney, the 
stroma consists of sparse, apparently inert, connective-tissue 
cells scattered among the renal tubules and about the blood 
vessels. Under pathological conditions this tissue in man may 
proliferate intensively (nephritis interstitialis). The appearance 
of bone, bone-marrow, and blood formation following special 
experimental conditions has been described in the kidney of the 
adult rabbit by Sacerdotti and Frattin (’02), by Poscharissky 
(’05), and Maximov (’07). The formation of bone and bone- 
marrow was interpreted by these authors as a local metaplasia 
of the connective-tissue cells of the stroma. According to 
Maximov, however, the blood formation observed in these 
experiments is to be interpreted as a local proliferation of young 
blood cells brought into the kidney from other hemopoietic 
centers by way of the blood current. In the opinion of Maximov, 
no one can seriously entertain a belief in the possibility of hemo¬ 
poietic potency in the scanty connective tissue of the adult 
kidney. 

Only in lower forms is the kidney regularly a seat of hemo¬ 
poiesis—the mesonephros of fishes being a permanent organ of 
blood formation. The meso- and metanephros of birds and 
mammals never display hemopoietic activity either in embryonic 
development or in the normal adult condition. However, it 
was shown by Danchakoff that in chick embryos the stroma of 
both the meso- and metanephros possesses hemopoietic potency, 
although under normal conditions this is not revealed. A marked 


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METANEPHRIC ANEAGE OF CHICK 


411 


granuloblastic transformation of the loose embryonic mesen¬ 
chyme in chick embryos was experimentally produced, the stroma 
of the meso- and metanephros being included in the general 
reaction. The development and differentiation of the nephric 
tubules in these embryos proceeded, however, in the usual way 
without any apparent disturbance. New nephric tubules con¬ 
tinued to form, but were separated by strands of granuloblastic 
tissue. In the metanephros the granuloblastic metaplasia was 
less marked than in the mesonephros. Contrary to what was 
observed in the mesonephros, comparatively few mobile cells 
developed from the apparently uniform mesenchyme of the 
metanephrogenic cord. The question as to whether the nephro¬ 
genic tissue consists of two substantially different tissues (an- 
lages of renal epithelium and stroma) appearing under the 
aspect of morphologically identical cells, was left undecided 
by Danchakoff: 

It is true, that the factors for the granuloblastic differentiation of 
the mesenchymal stroma become, under the present experimental 
conditions, so powerful as to transform a large amount of mesenchymal 
cells into granuloblasts even in a region of the organism in which 
granulopoiesis has never occurred in a typical development. It would, 
therefore, be only natural to expect that, if the nephrogenic tissue proper 
possessed a potentiality for granuloblastic differentiation, it would 
have manifested it under the present condition. The granuloblastic 
potentiality may, however, still be a property of the nephrogenic tissue, 
but the factors for the specific organization of the nephrogenic tissue 
into the renal epithelial tubules, which are unknown to us, may be of a 
more decisive nature for their realization. 

More information concerning the development and differ¬ 
entiation of the nephrogenic anlage may be obtained by a study 
of the development of the anlage isolated from its normal en¬ 
vironment and allowed to grow outside of the organism. 
Champy studied in detail the behavior of adult and embryonic 
kidney tissue outside the organism. Using Harrison’s tissue 
culture method as modified by Burrows and Carrel, he observed 
first an intensive proliferation of the cells of the renal tubules, 
followed by disintegration of the tubules to form cords of epithe¬ 
lial cells. According to his observations, the cords of epithelial 


412 


RUTH RAND ATTERBURY 


cells later broke down, their cells intermingling with those of the 
connective tissue to form an undifferentiated mesenchymal 
tissue. His conclusions are somewhat surprising. A fully 
differentiated kidney epithelium exercising in the organism a 
highly specialized function, if transplanted into an artificial 
culture medium, not only continues to live, but shows itself 
capable of a series of reversible reactions which bring it back 
to its undifferentiated state. 

Uninterrupted growth of tissue for a considerable length of 
time is not conveniently obtained by the tissue-culture method. 
New data concerning the developmental potencies of the 
nephrogenic tissue might be secured if the nephrogenic anlage 
were isolated from its normal relations within the organism and 
transplanted into a milieu which would permit of its undisturbed 
growth throughout a considerable period of time. At the sug¬ 
gestion of Doctor Danchakoff, I undertook the problem of 
studying the course of development of the metanephric anlage 
transplanted into the allantois of the chick embryo. The 
metanephric anlage of a chick of seven days’ incubation was 
chosen because, 1) at this stage the process of differentiation, 
though started, has not proceeded very far (for example, the 
Malpighian corpuscles are entirely lacking), and, 2) at this stage 
it is relatively easy to separate the metanephric anlage from the 
surrounding organs. 

In this study special attention has been given to the gradual 
differentiation of the specific structures of the, kidney from the 
morphologically homogeneous mesenchyme, which at the time 
of grafting forms the greatest part of the metanephric anlage. 
The development of the stroma has also been studied in detail 
especially with reference to its hemopoietic potency. 

I wish to express my gratitude to Doctor Danchakoff, to 
whom I owe the problem, for her constant help and kind en¬ 
couragement in this work. 


METANEPHRIC ANLAGE OF CHICK 


413 


MATERIAL AND METHOD 

The material upon which this investigation is based consists 
for the most part of grafts of the metanephric anlage isolated 
from chick embryos at a stage of seven days’ incubation and 
grown on the allantois of seven-day chick embryos. The eggs 
were opened under aseptic conditions and the embryos laid out 
in sterilized glass dishes. An opening was made along the 
ventral midline, and the heart, liver, and digestive tract were 
cut away. The glands and ducts of the urogenital system were 
thus exposed. Before attempting to isolate the metanephric 
anlage, it was found most convenient to remove the whole urogeni¬ 
tal system from the body cavity and to transfer it to a watch- 
glass, disturbing the general relations as little as possible. Thus 
the metanephric anlage in relation to the various parts of the 
urogenital system could more readily be seen and dissected out. 
The metanephric anlage was separated out by cutting the ure¬ 
teric diverticulum just above its entrance into the cloaca and by 
teasing the anlage apart from its connections with other tissues. 
The organ at this stage is about 1 mm. in length. Care was 
taken to keep the tissues well moistened with blood plasma dur¬ 
ing the process of isolation. Text figure A is a diagram of the 
separated metanephros as it appeared under the binocular 
microscope. The figure shows that the metanephric diverticu¬ 
lum has already begun to branch. The branches are arranged 
in two groups, a cranial, larger group, and a caudal, smaller 
group, joined together by an unbranched portion of the ureteric 
channel. 

The eggs used as hosts for the grafts were likewise at a stage 
of seven days’ incubation. At this time the allantois is well 
supplied with a vascular net and conditions are extremely 
favorable for the establishment of vascular connections be¬ 
tween the graft and the host. The method of procedure in the 
grafting was that used and described by Danchakoff (T6). 
A window was sawed out over a large allantoic vessel, upon which 
the metanephric anlage was carefully laid. In some cases the 
entire metanephric anlage was grafted, in other cases only its 


414 


RUTH RAND ATTERBURY 


cranial or its caudal part. The window was closed and sealed 
with paraffin and the egg returned to the incubator. The 
grafts were permitted to grow through various stages ranging 
from one to twelve days. Helly’s fluid was used for fixation and 
celloidin for embedding. The blocks were cut in serial sections 
and stained in eosin-azur. 

OBSERVATIONS 

A. Structure of the metanephric anlage at the time of grafting 

In order to determine the extent of growth and differentiation 
of the metanephric anlage in the allantois, a careful study was 
made of the structure which the anlage presented at the time 
of grafting. As seen in text figure A, the ureteric bud at this 
stage has already grown into the tissue of the metanephrogenic 
strand and has given rise to two groups of short branches, a 
cranial group and a caudal group. These branches constitute 
the primordia of the collecting tubules of the kidney. The 
ureteric branches of the caudal group are apparently in a slightly 
more advanced stage of development than the cranial. They 
seem somewhat longer and in several instances they have begun 
to give off secondary branches. Figure 1 is a photograph from 
a section of the metanephric anlage of a seven-day chick embryo 
and illustrates the extent of differentiation at this stage. The 
figure represents a section from the caudal, and, therefore, 
the most highly differentiated, part of the anlage. The ureter, 
cut longitudinally, appears in the form of a tubule with a dis¬ 
tinct lumen. This tubule has given off a primary ureteric branch 
which has started a further dichotomous division, forming in 
this way secondary branches. The lumen of both the ureter 
and its derivative is lined with simple columnar epithelium. 
The nuclei of the epithelial cells are large and have distinct 
nucleoli. Mitotic figures at this stage are found in large numbers. 

Text figure A shows the branches of the metanephric divertic¬ 
ulum thickly covered with dense masses of cells. In sections 
these appear in the form of a mantle of mesenchymal cells in 
close apposition with the branches of the ureter bud. These cells 


METANEPHRIC ANLAGE OF CHICK 


415 


constitute the nephrogenous tissue proper. The cells in im¬ 
mediate contact with the ureteric epithelium appear somewhat 
hypertrophied. They have acquired an epithelial arrangement, 
grouping themselves radially around the ureteric epithelium. 
The cells at a distance from the ureteric branches are smaller 
and less densely packed. 

The accounts of the normal development of the metanephros 
usually describe the nephrogenous tissue as being divided into 
two zones-—an inner zone, the anlage of the secretory tubules 



Text figure A Diagrammatic drawing of metanephric analage, isolated from 
a seven-day chick embryo, as it appeared under dissecting microscope. H, 
cranial group of ureteric branches; N.T., nephrogenous tissue; T, caudal group of 
ureteric branches; U, unbranched portion of ureteric channel. 


proper, and an outer zone, the anlage of the connective-tissue 
capsule and stroma of the kidney. The outer zone is described 
as developing in advance of the inner zone and forming a rather 
broad investment around the organ. According to Felix, the 
two zones are particularly well defined in the sea-gull. But both 
Felix and Lillie agree that in the chick it is practically impossible 
to distinguish the two zones. As seen from figure 1, the two 
zones are indistinguishable in the anlage of the seven-day chick 
embryo. The denser tissue around the ureteric branches merges 




416 


RUTH RAND ATTERBURY 


gradually into the looser mesenchyme which invests the whole 
anlage and which also occupies all the interstices between the 
ureteric branches. 

B. Growth of the metanephric grafts in the allantois 

It may be stated at once that both the ureter bud and the 
nephrogenous tissue proper survive and grow when grafted on 
the allantois. Indeed, the metanephric anlage of a seven-day 
chick embryo seems as fully capable of growth and differentiation 
in the allantois as in its normal environment within the organism. 
Figures 2 and 7 represent the metanephric anlage after seven and 
ten days’ growth, respectively, and illustrate to what extent 
growth and differentiation may proceed. Figure 2 is remarkable 
in showing that the metanephric anlage has developed in the 
form of two separate lobes, a larger and a smaller one. These 
two lobes, joined together by the main ureteric channel, have 
developed evidently from the cranial and caudal groups of ureteric 
branches present in the anlage at the time of grafting (text figure 
A). Figure 7 represents a section through a metanephros, the 
development and differentiation of which took place in the 
allantois. Almost all of this tissue has developed during the 
ten days’ growth in the allantois. The tiny metanephric anlage, 
which at the time of grafting was scarcely the size of a pin¬ 
head, has been transformed into a large, highly complex, lobu- 
lated organ very similar to the normal kidney of a chick embryo 
of corresponding age (seventeen days). 

The process of growth and differentiation of the metanephric 
anlage in the allantois was carefully studied and compared 
with the development of an undisturbed anlage at corresponding 
stages. For sake of clearness these observations will be recorded 
under separate headings: 1) Growth of the ureteric epithelium; 
2) Growth and differentiation of the nephric epithelium; 3) 
Development of the stroma. 

1 . Growth of the ureteric epithelium. The early stages of devel¬ 
opment of the metanephric structures in the allantois can be 
more easily followed by the study of the growth of the cranial 


METANEPHRIC ANLAGE OF CHICK 


417 


part of the anlage, which, at the time of grafting, is slightly less 
advanced in the process of differentiation. Just as in typical 
development, the primary ureteric branches grow in the allantois 
and carry with them parts of the nephrogenous tissue which 
closely cap their somewhat expanded ends. After the branches 
have attained a certain length, dichotomous division takes place 
at their blind terminations, giving rise to secondary tubules. 
This usually occurs at the end of the first or in the early part of 
the second day of growth in the allantois. Figure 3 represents 
a section through a graft of the metanephric anlage after twenty- 
four hours’ growth in the allantois and illustrates well the branch¬ 
ing of a primary ureteric tubule and the development of the 
secondary tubules. The primary tubule with its forked end 
presents an appearance very like the letter Y. The wall of the 
ureteric tubule is formed by simple, high columnar epithelium 
with large nuclei and deeply basophilic cytoplasm. The nuclei 
contain a rich chromatin network and one or more well-defined 
nucleoli. At the forking of the branches may be seen a large 
cell in mitosis. 

The ureteric branches of the second order continue to grow, 
their- cells proliferating by mitotic division. By the end of the 
second day in the allantois they in turn give rise to tertiary 
tubules. By further dichotomous division at the blind end of 
the newly formed tubules the ureteric tree grows and becomes 
more and more complex. Figure 4 shows the structure of the 
metanephros after two days’ growth in the allantois. A second¬ 
ary ureteric tubule ( 2') is seen dividing into two tertiary tubules 
(S'). These have grown to a considerable length. At the blind 
end of the tertiary tubule (S') a new subdivision is taking place, 
giving rise to tubules of the fourth order (4').' Subdivision of 
the primary ureteric branches goes on repeatedly in this manner, 
the branches so formed corresponding to the collecting tubules 
of the normal kidney. Each primary ureteric branch forms in 
this way a unit, the starting-point of a ureteric tree. The 
various lobules of the metanephric graft pictured in figure 7 
correspond to these units, each lobule constituting a primary 
ureteric tree. As seen from the study of the growth of the 


418 


RUTH RAND ATTERBURY 


ureteric epithelium in the allantois, the formation of the collecting 
tubules in the grafts does not differ from that usually described 
for their normal development within the organism. 

On the other hand, the study of the ureteric epithelium in the 
allantois reveals in it a potency which is not exhibited under 
normal conditions, i.e., a phagocytic digestive capacity. As 
might be expected, extirpation of the metanephric anlage from 



Text figure B Camera-lucida drawing to illustrate phagocytic activity of 
ureteric epithelium against degenerating red corpuscles. Metanephric graft of 
three days’ growth. B.C., Bowman’s capsule; c., capillary; D.E., degenerating 
erythrocytes; G, developing glomerulus; I.E., ingested erythrocyte; I. Hb-c., 
ingested hemoglobin-containing particle; N.T., nephrogenous tissue; U.T., 
large collecting tubules. 

the body cavity of the embryo is usually accompanied by hemor¬ 
rhage. It is not uncommon, therefore, in the grafts to find red blood 
corpuscles present in the lumen of the ureter bud. This new 
environment is apparently unfavorable for their maintenance and 
they gradually undergo degeneration. When degenerating eryth¬ 
rocytes are present within its lumen, the ureteric epithelium 
is seen to exhibit an intensive phagocytic activity. Text figure B 






METANEPHRIC ANLAGE OF CHICK 


419 


shows a cross-section through a tubule filled with a mass of 
degenerating erythrocytes. Numerous epithelial cells lining the 
tubule are seen with cytoplasmic processes directed toward the 
mass of erythrocytes. In the lower part of the tubule may be 
seen actually ingested pieces of hemoglobin-containing cyto¬ 
plasm (I . Hb.-c .). Above, the ureteric epithelium has ingested 
an erythrocyte, the nucleus of which has not yet disintegrated. 
Phagocytic activity by kidney cells of the frog has been described 
by Smallwood (’08) inacaseof hemorrhagic kidney. He observed 
red blood corpuscles in the lumen of tubules and particles of 
hemoglobin-containing cytoplasm within the epithelium. Small¬ 
wood does not state in what kind of epithelial cells he found the 
ingested material, but judging from his figures it must have been 
the ureteric epithelium of large collecting tubules. The presence 
of degenerating corpuscles in the lumen of the ureteric tubules 
is frequent in the early stages of kidney growth in the allantois, 
but they are never found in grafts of six or more days’ growth. 
One might conclude, therefore, that the process of digestion of 
the erythrocytes present in the tubules has been completed by 
this time. 

2. Growth of the nephric epithelium. As stated above, the 
nephrogenous tissue at the time of grafting consists of a dense 
mass of mesenchymal cells clustering thickly about the branches 
of the ureteric diverticulum. This tissue likewise survives and 
grows in the allantois. It reaches a high degree of differentiation 
in the formation of typical nephric tubules. 

In figure 3 the nephrogenous tissue appears as a continuous 
mantle of cells covering the forked terminations of the primary 
ureteric branch. The cells in immediate contact with the ure¬ 
teric epithelium are thickly packed and arranged vertically with 
respect to the ureteric epithelium; the outer, more distal cells, 
are looser and are grouped concentrically about the more proxi¬ 
mal cells. This arrangement of the nephrogenous tissue cor¬ 
responds to that present in the anlage at the time of grafting 

(fig- !)• 

As may be seen in figure 3, several structural differences exist 
between the proximal and distal cells of the nenhrogenous tissue. 


THE AMERICAN JOURNAL OF ANATOMv. VOL. 31. NO. 4 


420 


RUTH RAND ATTERBURY 


The outer cells differ but slightly from the loose mesenchymal 
cells of the allantois. A number of them appear in the form of 
round, mobile cells. Their nuclei are large and vesicular with 
well-defined nucleoli. Their cytoplasm forms a narrow rim 
around the nucleus and appears to be more basophilic than that 
of the allantoic mesenchyme. The cell bodies are practically the 
same size as those of the surrounding mesenchyme. The cells 
in immediate proximity with the ureteric epithelium, on the other 
hand, have markedly hypertrophied—in some cases they appear 
two and three times the size of the more distal cells. The nuclei 
have become large and oval, containing prominent nucleoli; 
the cell bodies appear heavy and deeply basophilic. A difference 
in the metabolism of the two kinds of cells must have found its 
expression in this difference of structure. 

With the growth of the ureteric tubules of the second order, 
the continuity of the nephrogenous mantle is broken and the 
nephrogenous tissue becomes split into separate masses. Part 
is carried forward on the blind terminations of the secondary 
tubules; part remains behind and condenses to form compressed 
spherical cell masses in the angles between the primary and 
secondary ureteric branches and between the two secondary 
branches. Figure 4 shows the nephrogenous tissue split into four 
separate cell masses. In this way the continuous mantle of 
nephrogenous tissue, such as that illustrated in figure 3, becomes 
split into two caps of tissue covering the blind ends of the newly 
formed tubules and into a number of small islands, which remain 
in close relation with the primary ureteric branch. These islands 
of nephrogenous tissue are transformed into metanephric spheres 
similar to those of normal development. The close association 
between the metanephric spheres and the branches of the ureter 
bud is a constant one. In the grafts no metanephric sphere is 
found except in close relation with a ureteric tubule. The split¬ 
ting up of the nephrogenous tissue proceeds hand in hand with 
the division and subdivision of the ureteric branches. The 
process is not dissimilar from that usually described for the 
typical development within the embryo. 


METANEPHRIC ANLAGE OF CHICK 


421 


The uriniferous tubules develop out of the separate meta- 
nephric spheres. Figure 5 represents a section through a graft 
of the metanephric anlage after three days’ growth in the allantois. 
Figure 6 represents another section through the same graft. 
Both photographs illustrate well the growth and differentiation 
ol the nephric tubules in grafts at early stages. In figure 5 the 
nephrogenous tissue is seen split up into numerous small cell 
masses, the metanephric spheres. In some of these the process 
of differentiation is well started. Each cell mass is closely as¬ 
sociated with a collecting tubule-—a branch of the ureter bud. 



Text figure C Camera-lucida drawing to illustrate formation of lumen. 
Metanephric graft of four days’ growth, c, capillary; M.S., metanephric sphere; 
Ms.c., mesenchymal cell; U.T., collecting tubule. 


This relationship is not clear in figure 5 for every cell mass, but 
the study of the graft in serial sections readily proves that each 
cell mass without exception is in close association with a ureteric 
tubule. In these preparations ureteric tubules are easily dis¬ 
tinguished from uriniferous tubules by their lighter stain. 

At first the cells forming the metanephric spheres bear no 
definite relation to each other, but are heaped together in a 
haphazard fashion; for example, as in the cell mass ( M.S .) in 
figure 4. During the second day’s growth in the allantois these 
cells begin to group themselves radially about a point near the 


422 


RUTH RAND ATTERBURY 


center of the metanephric sphere, the cells nearest the center 
acquiring a distinct epithelial arrangement. This stage is well 
illustrated in figure 5 ( M.S .), and in figure 6 (. M.S .). The central 
cells resemble the proximal, epithelially arranged cells of the 
nephrogenous mantle described in figure 3. They appear larger 
than the more peripheral cells of the cell mass with large oval 
nuclei. During this phase of development the cells are deeply 
basophilic. Soon, however, the central, radially arranged, 
epithelial-like cells undergo a change. At their pointed central 
ends the cytoplasm begins to take a faintly pinkish stain—a 
condition which might be considered a precursor of secretory 
activity. The central pointed ends are then flattened and pushed 
apart by the appearance of an acid-staining fluid at the point 
upon which the radially arranged cells had previously converged. 
Text figure C is a camera-lucida drawing illustrating this phenom¬ 
enon. A distinct lumen is formed in later stages. It seems, 
therefore, that the formation of a lumen may be due to the prod¬ 
ucts of secretory activity of the epithelial-like cells, which 
force the appearance of a space in the center of the metanephric 
sphere and subsequently its gradual widening. An analogous 
process was observed during the formation of the lumen in the 
normally developing metanephros of a nine-day chick embryo. 

In this manner the metanephric sphere is converted into a 
thick-walled metanephric vesicle. A metanephric vesicle is 
found in figure 6 (M.V.). The vesicle grows, its lumen widens, 
and the whole structure appears to develop distally from the 
collecting tubules with which it is associated. The growth of the 
vesicle takes place not so much through mitotic division of its 
cells, as by addition of cells from the more peripheral parts of 
the sphere. Mitotic figures at this stage of development are 
not as numerous as one would expect. The outermost cells of 
the sphere, too, eventually seem to contribute to the formation 
of the nephric tubules. They gradually become exhausted, 
only a few cells remaining behind in the form of scattered mesen¬ 
chymal cells situated among the developing renal tubules. The 
sharp distinction of the nephrogenous tissue into inner and 
outer zones, therefore, does not seem to be supported by these 
observations. 



METANEPHRIC ANLAGE OF CHICK 


423 


4 he vesicles continue to grow and shortly acquire a triple 
bend forming the S-shaped structure so characteristic of the 
normally developing metanephros. Soon the blind end of the 
S-shaped tubule is seen in contact with the adjacent ureteric 
tubule. An opening is then established, the cells of the separat¬ 
ing membrane being pushed aside and rearranged. In this way 
the nephric epithelium becomes continuous with the ureteric 
epithelium. From this time on growth of the tubule proceeds 
by mitotic division of the epithelial cells. 

Figure 5 shows an S-shaped tubule (*S). Its structure differs 
in no respect from the S-shaped tubule of the normal metanephros. 
The section following that from which figure 5 has been photo¬ 
graphed shows that this tubule has already acquired an opening 
into the collecting tubule (l .T.). Just as in the normally develop¬ 
ing uriniferous tubule, the S-shaped tubule of the grafts may be 
divided into three parts, an upper limb, middle piece, and lower 
limb. The distal part of the lower limb gives rise to Bowman’s 
capsule. In the S-shaped tubule (S) of figure 5, the cells of 
the outer wall of the lower limb already appear flattened. The 
S-shaped tubule (S) of figure 6 is in a still more advanced stage 
of differentiation. The distal end of the lower limb has grown 
around the structure in the form of a crescent, enclosing the 
upper limb within its arc. The cells of the outer or parietal layer 
are simple, flattened, and polygonal. The cells of the inner 
or visceral layer are likewise simple, but large and columnar 
and of a peculiar elongated pear-shape. The narrow interval 
between the two layers constitutes the uriniferous chamber. 
The same figure shows a Bowman’s capsule ( B.C .) in a still 
later stage of differentiation. The capsule has increased in 
size, its crescentic form tending to become more spherical. The 
urinary chamber appears greatly distended-—a fact which might 
be taken as indirect evidence for the functional activity of the 
cells at this time. 

The process of differentiation of the metanephric anlage in 
the allantois proceeds still further, leading to the appearance of 
typical glomeruli—structures which were not present at the 
time of grafting and which indeed do not appear in the normal 



424 


RUTH RAND ATTERBURY 


metanephros until the ninth day of incubation. In the grafts 
the first signs of the glomeruli appear during the third day’s 
growth. Tiny allantoic capillaries grow into the Bowman’s 
capsules, forming the typical glomerular knots so characteristic 
of the normal development. Figure 6 shows well the ingrowth of 
the vessels into the Bowman’s capsules of the graft. The space 
between the Bowman’s capsule and the upper limb of the S- 
shaped tubule (S) described above is occupied by a mass of 
mesenchymal cells. A capillary filled with erythrocytes is seen 
growing into this mass (C). In the larger Bowman’s capsule 
(. B.C .) the vasculoconnective tissue appears greatly increased 
in amount. The capillary network is recognized by the presence 
of erythrocytes and a granular leucocyte. As a result of the 
process of differentiation described above, apparently normal 
Malpighian corpuscles are formed. 

After several days’ growth the metanephric anlage attains 
in the allantois a high degree of organization (figs. 2 and 7). 
Even under low power the tubules and Malpighian corpuscles 
are easily recognized. Huber (’17) has been able to identify the 
various segments of the uriniferous tubules of the adult fowl 
by isolating complete tubules through a process of maceration 
and teasing. It is possible to identify in sections from grafts 
of late stages segments of tubules corresponding to these de¬ 
scribed by Huber for the adult fowl. The secretory tubules are 
readily distinguishable from the collecting ones. The cytoplasm 
of the secreting epithelium is finely granular, and in eosin-azur 
preparations takes a pinkish stain. The cells are low columnar 
and somewhat pyramidal in shape. The segments of Henle’s 
loop appear in grafts of seven or more days’ growth, but never 
grow to their full extent. The collecting tubules, on the other 
hand, are characterized by clear, distinctly basophilic cytoplasm. 
Their cells are cuboidal, with round chromatic, centrally placed 
nuclei. The lumen of the collecting tubules seems wider than 
that of the secretory tubules. Text figures D and E are high- 
power camera-lucida drawings from grafts of six and eight days’ 
growth and show various sections of the Malpighian bodies, the 
secretory and collecting tubules of the grafts, as they appear 
at these stages. 


METANEPHRIC ANLAGE OF CHICK 


425 


3. Growth oj the stroma. During the early stages of growth 
in the allantois, the loose mesenchyme of the anlage develops 
a high degree of vascularization. The development of the vessels 
was leadily followed out. At the periphery of the anlage and 
later throughout its stroma spaces separating groups of individual 
cells are seen to appear. These spaces are apparently distended 
with fluid. The mesenchymal lining of the spaces, at first irreg¬ 
ular in its outline, soon acquires the character of typical endo- 


N.T. 


Ms. 


Text figure D Camera-lucida drawing to illustrate granulopoiesis and seg¬ 
ments of urinary tubules. Metanephric graft of six days’ growth, c., capillary; 
Gbl., granuloblast; Hbl., hemoblastjxlf.jB., Malpighian body; Ms.c., mesenchymal 
cell; N.T., secretory tubules; U.T., collecting tubule. 

thelium. The spaces establish communications with one 
another, forming in this way a vascular net, which soon estab¬ 
lishes connections with the allantoic vascular channels. Figure 
7 illustrates the high degree of vascularization which develops 
in grafts of the metanephric anlage. Broad vascular channels 
encircle the organ and traverse the spaces separating its various 
lobes. Smaller vessels and capillaries are numerous between 
the lobules and among the nephric tubules. 






RUTH RAND ATTERBURY 


426 

After the vascularization of the graft has become well de¬ 
veloped, changes may be observed in the scanty mesenchymal 
cells of the stroma. In typical development these cells form the 
connective tissue of the kidney. In the grafts they take a new 
line of differentiation, developing into small, scattered foci of 
granuloblastic tissue. 

The first signs of granuloblastic differentiation appear in 
grafts of six days’ growth. This process is illustrated by text 



Text figure E Camera-lucida drawing to illustrate granulopoiesis and various 
segments of urinary tubules. Metanephric graft of eight day’s growth. ci.H., 
ascending arm of Henle; B.C., Bowman’s capsule; c., capillary; d.H., descending 
arm of Henle; end., endothelium; e., erythrocyte; G., glomerulus; Gbl., granulo¬ 
blast; Get., granulocyte; N.T., convoluted tubules; U.T., collecting tubule. 

figure D, in which can be seen an early stage in the develop¬ 
ment of a granulopoietic focus. Several mesenchymal cells 
have apparently lost their syncytial arrangement and appear 
as free, hypertrophied, deeply basophilic, amoeboid cells. They 
exhibit all the characteristics of typical hemoblasts. A few of 
these cells already contain acidophilic granules in their cyto¬ 
plasm. In some the nucleus has become slightly polymorphic. 






METANEPHRIC ANLAGE OF CHICK 


427 


These granuloblasts are undoubtedly of local origin, their gradual 
development from the mesenchyme being easily followed. 

Text figure E illustrates the extent of the granuloblastic 
metaplasia which the stroma of the grafted anlage may attain. 
Heavy strands of granuloblasts fill in the spaces between the 
tubules. Granulopoiesis here appears in more advanced stages. 
Mature granulocytes with rod-shaped granules and lobulated 
nuclei have now developed. The intensive changes illustrated 
by this figure do not appear uniformly throughout the whole 
graft. In the central part of the lobules, where the tubules are 
closely packed together and the vascularization is scanty, only 
isolated granuloblasts can be found. A more or less intensive 
granulopoiesis, however, is regularly observed in regions where 
the stroma retains its loose texture and is well supplied by vascu¬ 
lar channels. 


CONCLUSIONS AND DISCUSSION 

1. The metanephric anlage of the seven-day chick embryo, 
when isolated and grafted on the allantois of a chick embryo of 
the same age, not only survives, but grows into a large organ 
and attains a high degree of differentiation.. 

The ureter bud, which at the time of grafting has already begun 
to branch, forms typical collecting tubules. This process is 
exactly similar to that usually described for the formation of the 
collecting tubules of the metanephros developing within the 
embryo. 

The nephrogenous tissue proper also continues its differentia¬ 
tion into the characteristic nephric vesicles. The nephrogenous 
cord, which at the time of grafting appeared in the form of a 
dense syncytium of apparently undifferentiated mesenchymal 
cells, proliferates and differentiates into a secretory epithelium. 
The formation of the lumen of the developing nephric vesicles 
appears to be the direct mechanical result of the secretory 
activity of the cells. The differentiation of the nephrogenous 
tissue leads to the appearance of typical glomeruli, structures 
which at the time of grafting were not present in the anlage. 
The growth and differentiation of the nephric tubules in the 


428 


RUTH RAND ATTERBURY 


grafts may reach a high degree of organization. In older grafts 
convoluted tubules and segments of Henle can be identified. 
The process of growth and differentiation of the secretory part 
of the kidney on the allantois does not differ from that which 
normally takes place in the undisturbed kidney anlage. 

The fact that the metanephric anlage of a seven-day chick 
embryo, when isolated from its normal relations within the 
organism and transplanted into the allantois, continues to 
differentiate into characteristic nephric tubules seems to indicate 
that at this time the young anlage has already become specifi¬ 
cally organized. Contrary to the results obtained by Champy, 
the parts of the anlage already differentiated (ureteric epithelium, 
epithelially arranged cells of the nephrogenous tissue) do not 
undergo a series of reversible changes, but show a marked 
capacity to retain their epithelial arrangement and to differentiate 
still further. Moreover, the apparently undifferentiated part 
of the anlage, which at the time of grafting appeared in the 
form of a dense syncytium of mesenchymal cells, showed the 
ability to acquire an epithelial arrangement and to form typical 
nephric tubules. The process of differentiation proceeds to such 
an extent that even the allantoic capillaries are induced to form 
t}^pical glomerular knots within the concavities of the 
Bowman’s capsules. The allantois seems to be as favorable a 
medium for the growth and differentiation of the metanephric 
anlage at this stage as its normal environment within the or¬ 
ganism. The grafted anlage may attain the same general form, 
size, and structure as that of the normal kidney of corresponding 
age. 

Not only does the transplanted metanephros acquire the 
structure of the normally developing kidney, it also, as far as 
histological evidence can determine, develops physiological 
activity. This development of both structure and function 
in the grafted anlage takes place despite the fact that it can be of 
no possible use to the general organism, since there is no outlet 
in the transplanted metanephros whereby the graft can get rid of 
the products of its secretory activity. Champy has ascribed the 
process of dedifferentiation of the metanephros in his cultures to 


METANEPHRIC ANLAGE OF CHICK 


429 


lack of functional stimulus. But, despite lack of functional 
stimulus, cultures of the metanephros in the allantois attain a 
high degree of differentiation. It is possible that the different 
results obtained by Champy and myself may be due to a dif¬ 
ference in the milieu in which the cultures were planted. A 
tissue possessing the potency will differentiate into nephric 
tubules, provided the conditions for its growth and differentiation 
are favorable. Under other conditions, this process may not 
take place, and even a tissue already specialized may lose its 
specific structures, as Champy seems to have shown. 

2. A digestive power is revealed by the ureteric epithelium 
which may be seen to ingest and digest red blood corpuscles. 

Unimportant as it may seem, the observation on the phagocytic 
and digestive activity displayed by the epithelial cells of the 
collecting tubules may prove of interest in view of the vast role 
of digestive activity recently attributed to the general mesen¬ 
chyme (Danchakoff, ’21). This power of digestion may sometime 
in the future be recognized as an active and simple agent in the 
so-called defenses of the organism. In this particular case a 
specialized mesodermal cell, the epithelial cell of the collecting 
tubules, exercises phagocytic and digestive activity. Other 
apparently specialized cells in the organism, for example, endo¬ 
thelial and mesothelial cells, although in the form of epithelial 
membranes, retain and exercise to a considerable extent the 
fundamental biological property of digestion. 

3. The stroma of the grafted anlage develops along lines 
different from that characteristic of its normal development. 
Normally this tissue gives rise to sparse connective-tissue cells; 
in the grafts it is transformed into large, scattered foci of granu- 
loblastic tissue. The granuloblastic activity displayed by the 
loose mesenchyme of the metanephric anlage in these grafts 
affords additional evidence for the equivalence of the mesenchyme 
and for its polyvalency in the different parts of the organism. 
In this instance the granuloblastic differentiation of the 
mesenchyme is the more interesting since, of all regions of the 
embryo’s body, the mesenchyme of the metanephros has shown 
itself the least responsive to this change after grafts of adult 


430 


RUTH RAND ATTERBURY 


splenic tissue. The granuloblastic differentiation of mesen¬ 
chymal cells is once more shown to be dependent upon environ¬ 
mental conditions. 

LITERATURE CITED 

Carrel, A., and Burrows, M. 1911 Cultivation of tissues in vitro. Journ. 
Exper. Med., vol. 13, pp. 387-396. 

Champy, C. 1913 La differenciation des tissus cultures en dehors de l’organ- 
isme. Bibl. Anat., T. 23, p. 184. 

1913 Sur les phenomenes cytologiques qui s’observent dans les 
tissus cultures en dehors de Lorganisme. Tissus epitheliaux et gland- 
ulaires. Compt. rend. soc. biol. Paris, T. 72, p. 987. 

1914 Quelques resultats de la methode des cultures de tissus, le rein. 
Arch, de Zool. Exper. et Gen., T. 54, pp. 307-386. 

Danchakoff, Vera 1916 The wandering cells in the loose connective tissue of 
the bird and their origin. Anat. Rec., vol. 10, pp. 483-492. 

1916 The differentiation of cells as a criterion for cell differentiation 
considered in relation to the small cortical cells of the thymus. Journ. 
Exper. Med., vol. 24, pp. 87-105. 

1916 Equivalence of different haematopoietic anlages (by method 
of stimulation of their stem cells). I. Spleen. Am. Jour. Anat., vol. 
20, pp. 255-308. 

1918 Equivalence of different haematopoietic anlages (by method of 
stimulation of their stem cells). II. Grafts of adult spleen on the 
allantois and response of the allantoic tissues. Am. Jour. Anat., 
vol. 24, pp. 127-173. 

1920 Immunity and the power of digestion. Bull. Marine Biol. 
Lab., vol. 38, p. 202. 

1920 Myeloid metaplasia of the embryonic mesenchyme in relation to 
potentialities and differential factors. Contrib. no. 49, Carnegie 
Institution of Washington. 

1921 Digestive activity of mesenchyme. A. The Ehrlich sarcoma 
cells as object. Am. Jour. Anat., vol. 29, pp. 431-489. 

Felix, N., and Buehler, A. 1906 Die Entwickelung der Harn und Ge- 
schlechtsorgane. Handbuch der Vergleichender u. experimentaler 
Entwickelungslehre der Wirbeltiere. Herausgegeben von Oskar 
Hertwig, Bd. 3, S. 81. 

Harrison, Ross G. 1904 An experimental study of the relation of the nervous 
system to the developing musculature in the embryo of the frog. Am. 
Jour. Anat., vol. 3. 

Huber, G. Carl 1917 On the morphology of the renal tubules of vertebrates. 
Anat. Rec., vol. 13, pp. 305-339. 

Huntington, George S. 1914 The development of the mammalian jugular 
lymph sac of the tributary primitive ulnar lymphatic and of the thoracic 
ducts from the viewpoint of recent investigations of the vertebrate 
lymphatic ontogeny, together with a consideration of the genetic rela¬ 
tions of the lymphatic and haemal vascular channels in the embryos of 
amniotes. Am. Jour. Anat., vol. 16, pp. 259-316. 


METANEPHRIC ANLAGE OF CHICK 


431 


Janosik, J. 1907 Ueber die Entwickelung der Nachniere bei den Amnioten. 

Arch. f. Mikr. Anat. u. Entw., Bd. 70, S. 23-82. 

Lillie, Frank R. 1908 The development of the chick. Henry Holt & Co. 
Maximov, A. 1907 Experimentelle Untersuchungen zur post-foetalen Histo- 
genese des myeloiden Gewebes. Zieg. Beitr., Bd. 41, S. 112-16G. 
Poscharissky, J. F. 1905 Ueber heteroplastiche Enoch enbildung. Zieg. 
Beitr., Bd. 38, S. 135-176. 

Sacerdotti, C., and Frattin, G. 1902 Ueber die heteroplastische Knochen- 
bildung. Virch. Arch., Bd. 168, S. 431-443. 

Smallwood, N. M. 1908 The kidney cells of the frog in phagocytic role. Anat. 
Anz., Bd. 32, S. 201-205. 

Uhlenhuth, E. 1912 Die transplantation des Amphibiensauges. Arch. f. 
Entw. des. Org., Bd. 33, S. 723-747. 

1915 The form of the epithelial cells in cultures of the frog skin and 
its relation to the consistency of the medium. Jour. Exper. Med., 
vol. 22, pp. 76-104. 


» 



PLATE 1 

EXPLANATION OF FIGURES 

1 Photograph of metanephric anlage of seven-day chick embryo to illustrate 
stage of differentiation at time of grafting. The ureter bud has given off primary 
branches, which in turn have begun dichotomous division to form secondary 
branches. The ureteric branches are capped with a dense mantle of nephro¬ 
genous tissue. 

2 Photograph from section of graft of metanephric anlage after seven 
days’ growth in the allantois. 

3 Photograph from a graft of the metanephric anlage after twenty-four 
hours’ growth in the allantois. A primary ureteric tubule has begun dichotomous 
division forming ureteric tubules of the second order. The ureteric epithelium is 
covered with a continuous mantle of nephrogenous tissue. 

4 Photograph from a graft of the metanephric anlage after two days’ growth 
in the allantois. To illustrate branching of ureteric tubules and splitting of 
nephrogenous tissue. M.S., metanephric sphere; 2', ureteric tubule of second 
order; 2', ureteric tubule of third order; 4', early stage in dichotomous division 
to form ureteric tubule of fourth order. 


432 


METANEPHRIC ANLAGE OF CHICK 

RUTH RAND ATTERBURY 


PLATE 1 



433 





PLATE 2 


EXPLANATION OF FIGURES 

5 Photograph from a graft of the metanephric anlage after three days’ growth 
in the allantois. To illustrate early stages in development of nephric tubules. 
M.S., metanephric sphere with central cells radially arranged; S, S-shaped 
tubule; U.T., ureteric tubules. 

6 Photograph from a graft of the metanephric anlage after three days’ growth 
in the allantois. To illustrate development of Malpighian corpuscles. B.C., 
Bowman’s chamber; c., capillary; G., developing glomerulus; M.S., metanephric 
sphere; M.Y., metanephric vesicle; S., S-shaped tubule with distal end differ¬ 
entiating to form Bowman’s capsule; U.T., collecting tubule. 


434 



PLATE 2 


METANEPHRIC ANLAGE OF CHICK 

RUTH RAND ATTERBURY 



435 




PLATE 3 

EXPLANATION OF FIGURE 

7 Photograph from a graft of the metanephric anlage isolated from a seven- 
day chick embryo and grown ten days in the allantois. To illustrate extent of 
growth and differentiation which metanephric anlage may attain. 


436 


metanephric anlage of chick 

RUTH RAND ATTERBURY 


PLATE 3 



437 












VITA 


1. Date of Birth: May 23, 1895. 

2. Place of Birth: New York City. 

3. Educational Institutions Attended: 

Wellesley College, Wellesley, Massachusetts, 1912-1916. 
Cornell University, Ithaca, New York, 1916-1918. 

Cornell Medical School, New York City, 1918-1919. 
College of Physicians and Surgeons, Columbia University, 
New York City, 1919-1922. 

4. Degrees: 

Bachelor of Arts, Wellesley College, 1916. 

Master of Arts, Cornell University, 1917. 

5. Honors: 

Durant Scholar (Junior and Senior), Wellesley College, 
1915-1916. 

Sigma XI, Cornell University, 1917. 

6. Publications: 

(1) On the Relation of the Head Chorda to the Pharyn¬ 

geal Epithelium in the Pig Embryo. A Contribution 
to the Development of the Bursa Pharyngea and the 
Tonsilla Pharyngea. 

Anat. Record, Vol. 13, pp. 465-491, 1917. 

(2) Bursa and Tonsilla Pharyngea. A Note on the Rela¬ 

tions in the Embryo Calf. 

Anat. Record, Vol. 16, pp. 251-264, 1919. 

(3) Potentialities of Different Parts in the Kidney Anlage. 

Abstr. Proc. Anat. Record, Vol. 18, p. 219, 1920. 


























































































































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